Cellular genomic DNA is susceptible to damage by several factors including UV light, radiation, toxins, chemicals, viruses, oxidation, and other endogenous and environmental damage. Cells respond to this damage through processes known as the DNA damage response (DDR) that identify and correct damage to the DNA based on the type of damage inflicted. Some types of damage can be chemically reversed (e.g., methylation and covalent bonds between adjacent pyrimidine bases). In contrast, single-strand breaks (SSBs) and double-strand breaks (DSBs) require restoration of the broken phosphodiester bonds.
In SSD, only one of the two strands of DNA incurs damage, so the complementary strand can be used as a template to correct the damage using an excision repair mechanism (e.g., base excision repair (BER), and nucleotide excision repair (NER). DSB damage is repaired using an end joining (EJ) mechanism (e.g., non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ)); or by a homologous recombination (HR) mechanism (e.g., via homology-directed repair (HDR)). Each of these DSB repair pathways can be prone to mistakes, which can cause deletion, insertion, rearrangement, frame-shift or other mutations that can lead to malignancy or other pathologies.
DSB repair pathways show a varying propensity for genetic loss. A relatively precise form of repair is homology-directed repair (HDR) that uses the identical sister chromatid as a template for Rad51-mediated strand invasion and nascent DNA synthesis. In contrast, EJ pathways are variably mutagenic, depending on the extent of end-processing and the fidelity of end-pairing. For instance, EJ via the NHEJ machinery used during V(D)J recombination (classical NHEJ) has the potential to be precise. However, if DSB ends are not readily ligated without prior significant processing (e.g., degradation of damaged bases or protruding single stranded DNA at the ends), such classical NHEJ can lead to deletion and/or insertion mutations. Furthermore, Ku-independent EJ (Alternative-NHEJ, Alt-NHEJ) often leads to deletion or insertion mutations, which are predominantly associated with short stretches of homology (microhomology) at repair junctions. Similar to Alt-NHEJ is the single-strand annealing (SSA) pathway of homologous recombination, which also causes deletions with homology at repair junctions, but involves extensive regions of homology. In addition, for each of these pathways, loss of correct end-pairing during the repair of multiple simultaneous DSBs can lead to chromosomal rearrangements. For instance, EJ between distal ends of two tandem DSBs (Distal-EJ) results in loss of the chromosomal segment between the DSBs.
The factors and pathways that influence DSB repair may be exploited for the purpose of developing experimental and therapeutic systems in a wide range of species. These systems may be used for various functions, such as to study gene function in plants and animals, to engineer transgenic cells and organisms, or to develop therapeutic interventions such as in vivo or ex vivo gene therapy in subjects with chromosomal aberrations or aberrantly expressed gene products. However, because these pathways are prone to cause different mutagenic outcomes, it is desired to develop systems that influence the DSB repair machinery to enhance the frequency of the desired outcome.